Characterization of Aberrantly Expressed MicroRNAs in Epstein-Barr Virus-associated Nasopharyngeal Carcinoma.

摘要

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) has been reported to be related to a number of genetic and epigenetic changes, however, the molecular mechanism leading to NPC tumorigenesis still remains unclear. Recently, microRNAs (miRNAs) have been demonstrated to play vital roles in NPC development via regulating cell proliferation, apoptosis, and cell migration and invasion. In this study, we aim to elucidate the role of miRNAs in NPC tumorigenesis in this study by identifying the miRNA aberration, investigating the possible functions of these aberrantly expressed miRNAs, and unraveling the role of stemness-related miRNAs in NPC cancer stem-like cells (CSCs).;By using Agilent Microarray with 866 human and 89 viral miRNA probes, miRNA expression profiles of multiple EBV-associated NPC tumor lines were generated. Compared to NP69, a nonmalignant nasopharyngeal epithelial cell line, 113 differentially expressed miRNAs were identified. Among the 58 down-regulated miRNAs in NPC, transcriptional silencing of miR-31 was consistently found in both NPC tumor lines and primary tumors. Down-regulation of miR-31 was detected in 6 of 7 (86%) EBV-positive tumor lines and 38 of 38 (100%) microdissected primary tumors, while all normal nasopharyngeal epithelia showed high expression of miR-31..;miR-31 is located at 0.5 Mb telomeric to CDKN2A (p16) on chromosome 9p21.3, which is commonly deleted in NPC. Homozygous deletion of both miR-31 and CDKN2A loci was confirmed in tumor lines X1915 and X99186. In the four tumor lines with intact miR-31, hypermethylation of 5' CpG islands was detected by methylation-specific PCR (MSP) and bisulfite sequencing analysis. Restoration of miR-31 transcription was demonstrated in the EBV-positive NPC cell line C666-1 treated with 5-aza-2'-deoxycytidine. These findings suggested that homozygous deletion and promoter hypermethylation are the major mechanisms for transcriptional silencing of miR-31 in NPC.;By microarray and bioinformatic analysis, a number of putative targets of miR-31 were identified. Among these candidates, FIH1 and MCM2 were found to be the targets of miR-31 in NPC. We have shown that binding of miR-31 on FIH1 and MCM2 mRNA 3'UTR suppressed their luciferase activity. Ectopic expression of miR-31 in NPC cells resulted in repression of FIH1 and MCM2 protein expression. Importantly, the restoration of miR-31 or knockdown of FIH1 expression significantly suppressed proliferation as well as migration of C666-1 cells. Clone-forming ability and anchorage-independent growth of C666-1 were significantly inhibited by miR-31 expression. Stably expressed miR-31 was also demonstrated to inhibit NPC tumor growth in nude mice. Furthermore, expression of p21 and phospho-p53 (Ser15) was found to be increased by FIH1 knockdown. These results implied that miR-31 is a critical NPC-associated miRNA which negatively regulates cell proliferation and migration via FIH1 repression.;By miRNA microarray analysis, we have screened for differentially expressed miRNAs in sphere-forming cells of EBV-associated NPC. In concordance with microarray findings, suppression of miR-96 and miR-183 in C666-1 spheroids was confirmed by qRT-PCR. Ectopic expression of miR-96 and miR-183 significantly reduced the sphere-forming and clone-forming ability of C666-1 cells. The findings implied that miR-96 and miR-183 repression is important in the formation of NPC CSCs.;In summary, several miRNAs were identified as potential tumor suppressor genes in NPC. miR-31 was found down-regulated by homozygous deletion or promoter hypermethylation in EBV-associated NPC. It plays roles in NPC pathogenesis by suppressing NPC cell proliferation, clone-forming ability, cell anchorage-independent growth, migration and in vivo tumor growth. Moreover, miR-96 and miR-183 were found to have a role in the maintenance of NPC stem-like properties. These findings suggested important tumor suppressive roles of miRNAs in regulating NPC tumorigenesis, and a better understanding on the miRNA mechanisms may potentiate better therapeutic strategies for NPC.